DNA topoisomerases I and II catalyze the breaking and rejoining of DNA strands in a way that allows the strands to pass through one another, thus altering the topology of DNA. Type I topoisomerases break a single DNA strand, while the type II enzymes break two strands of duplex DNA. Both enzymes can perform a variety of similar topological interconversions, including relaxation of super coiled DNA, knotting/unknotting and catenation/decatenation of duplex DNA.
Both topoisomerases I and II can provide the topological interconversions necessary for transcription and replication. For example, topoisomerase I can provide the necessary unlinking activity for efficient in vitro DNA replication (Minden, et al., Journal of Biological Chemistry, 260:9316, 1985; Nagata, et al., Proceedings of the National Academy of Sciences, U.S.A., 80:4266, 1983; Yang, et al., Proceedings of the National Academy of Sciences, U.S.A., 84:950, 1987) however, topoisomerase II can also facilitate the replication of SV40 DNA by HeLa cell lysates (Yang, et al., Proceedings of the National Academy of Sciences, U.S.A., 84:950, 1987). Genetic studies in yeast reveal that both replication and transcription proceed in single mutants deficient in either topoisomerase I or II (Goto, et al. Proceedings of the National Academy of Sciences, U.S.A., 82:7178, 1985; Uemura, et al., EMBO Journal, 3:1737, 1984; Thrash, et al., Proceedings of the National Academy of Sciences, U.S.A., 82:4374, 1985). In cells lacking both topoisomerases, transcription and replication are dramatically reduced (Uemura, et al., EMBO Journal, 5:1003, 1986; Brill, et al., Nature, 326:414, 1987).
Several lines of evidence suggest that topoisomerase I normally functions during transcription. The enzyme has been shown to be localized preferentially to actively transcribed loci by immunofluorescence (Fleishmann, et al., Proceedings of the National Academy of Sciences, U.S.A., 81:6958, 1984), and by co-immunoprecipitation with transcribed DNA (Gilmore, et al., Cell, 44:401, 1986; Muller, et al., EMBO Journal, 1237, 1985). Furthermore, topoisomerase I cleavage sites have been mapped to regions in and around transcribed DNA (Bonner, et al., Cell, 41:541, 1985; Gilmour, et al., Molecular Cell Biology, 7:141, 1987; Stewart, et al., Cell, 50:109, 1987). Nonetheless, at least in yeast, topoisomerase II can apparently substitute for the functions of topoisomerase I in transcription (Uemura, et al., EMBO Journal, 3:1737, 1984; Thrash, et al., ibid).
Of further interest is the use of topoisomerase I in classifying autoimmune disease. Autoimmune diseases are diseases in which an animal's immune system attacks its own tissues. Often the various types of autoimmune disease can be characterized based upon the specificity of autoantibodies which are produced. For example, it is well known that the serum of patients having the connective tissue autoimmune disease progressive systemic sclerosis (PSS), also known as scleroderma, frequently contain antibodies to such nuclear antigens as topoisomerase I. Thus, the ability to accurately detect the presence of antibodies reactive with topoisomerase I can greatly assist in evaluating the prognosis and planning, or monitoring, the appropriate therapy of patients with scleroderma.
Unfortunately, the existing commercial dectection systems utilize crude exacts of cell nuclei, usually of rabbit or bovine origin, which contain antigens other than topoisomerase I. As a result, these systems will detect antibodies which react with nuclear antigens other than topoisomerase I such as, for example, the centromere, and, as a result, may give false positive results which can lead to an incorrect diagnosis. Thus, there is considerable need for a system which detects only antibodies to topoisomerase I and not other eukaryotic peptides.